US20210222173A1 - Compositions and Methods for Lactate Dehydrogenase (LDHA) Gene Editing - Google Patents

Compositions and Methods for Lactate Dehydrogenase (LDHA) Gene Editing Download PDF

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US20210222173A1
US20210222173A1 US17/212,901 US202117212901A US2021222173A1 US 20210222173 A1 US20210222173 A1 US 20210222173A1 US 202117212901 A US202117212901 A US 202117212901A US 2021222173 A1 US2021222173 A1 US 2021222173A1
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Zachary William Dymek
Shobu Odate
Anette HUEBNER
Srijani Sridhar
Bradley Andrew Murray
Walter Strapps
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Intellia Therapeutics Inc
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Definitions

  • Oxalate normally eliminated in urine as waste by the kidneys, is elevated in subjects with hyperoxaluria.
  • hyperoxaluria There are several types of hyperoxaluria, including primary hyperoxaluria, oxalosis, enteric hyperoxaluria, and hyperoxaluria related to eating high-oxalate foods.
  • Excess oxalate can combine with calcium to form calcium oxalate in the kidney and other organs.
  • Deposits of calcium oxalate can produce widespread deposition of calcium oxalate (nephrocalcinosis) or formation of kidney and bladder stones (urolithiasis) and lead to kidney damage.
  • kidney complications in hyperoxaluria include blood in the urine (hematuria), urinary tract infections, kidney damage, and end-stage renal disease (ESRD). Over time, kidneys in patients with hyperoxaluria may begin to fail, and levels of oxalate may rise in the blood. Deposition of oxalate in tissues throughout the body, e.g., systemic oxalosis, may occur due to high blood levels of oxalate and can lead to complications in at least bone, heart, skin, and eye. Kidney failure can occur at any age, including in children, especially in subjects with hyperoxaluria. Renal dialysis or dual kidney/liver organ transplant as the only treatment options.
  • PH Primary hyperoxaluria
  • AGT/AGT1 alanine-glyoxylate aminotransferase
  • PH2 glyoxylate/hydroxypyruvate reductase, or GR/HPR
  • PH3 4-hydroxy-2-oxoglutarate aldolase, or HOGA.
  • mutations are found in the enzyme alanine glyoxylate aminotransferase (AGT or AGT1) that is encoded by the AGXT gene.
  • AGT converts glyoxylate into glycine in liver peroxisomes.
  • mutant AGT is unable to break down glyoxylate, and levels of glyoxylate and its metabolite oxalate increase.
  • Humans cannot oxidize oxalate, and high levels of oxalate in subjects with PH1 cause hyperoxaluria.
  • a 24-hour urine may be collected and the oxalate, glycolate, and other organic acid levels are measured. Genetic testing or liver biopsy can be performed for a definitive diagnosis of genetic forms of hyperoxaluria. See, e.g., Cochat P et al., (2012) Nephrol Dial Transplant 5:1729-36. In normal healthy subjects the 24-hour urine oxalate and glycolate levels are less than 45 mg/day but in hyperoxaluria patients, levels of urinary oxalate greater than 100 mg/day are typical. See, e.g., Cochat P. (2013). N Engl J Med 369:649-658.
  • Plasma glycolate levels in normal subjects are typically 4-8 micromolar but in hyperoxaluria patients glycolate levels can range widely and are elevated in 2 ⁇ 3rds of hyperoxaluria subjects. See, e.g., Marangella, M et al. (1992) J. Urol. 148:986-989. While most patients with genetic forms of hyperoxaluria are now diagnosed through genetic testing, a 24-hour urine test is the primary method used to follow hyperoxaluria subjects for treatment responses. Id.
  • Lactate dehydrogenase is an enzyme found in nearly every cell that regulates both the homeostasis of lactate and pyruvate, and of glyoxylate and oxalate metabolism.
  • LDH is comprised of 4 polypeptides that form a tetramer. Five isozymes of LDH differing in their subunit composition and tissue distribution have been identified. The two most common forms of LDH are the muscle (M) form encoded by the LDHA gene, and the heart (H) form encoded by LDHB gene. In the perioxisome of liver cells, LDH is the key enzyme responsible for converting glyoxalate to oxalate which is then secreted into the plasma and excreted by the kidneys. Lai et al. (2016) Mol Ther. 26(8):1983-1995.
  • LDHA siRNA directed to hepatocytes via conjugation with N-acetylgalactosamine (GalNAc) residues was used to mediate LDHA silencing in mouse models of hyperoxaluria. See, Lai et al. (2016) Mol Ther. 26(8):1983-1995.
  • Treatment of mice with this LDHA siRNA resulted in a reduction of hepatic LDH and efficient oxalate reduction and prevented calcium oxalate crystal deposition in both genetically engineered mouse models of hyperoxaluria and in chemically induced hyperoxaluria mouse models. Id. Suppression of hepatic LDH in mice did not result in acute elevation of circulating liver enzymes, lactate acidosis, or exertional myopathy.
  • LDHA siRNA treatment of both non-human primates and humanized chimeric mice in which the liver is comprised of up to 80% human hepatocytes. Id.
  • the disclosure provides compositions and methods using a guide RNA with an RNA-guided DNA binding agent such as the CRISPR/Cas system to substantially reduce or knockout expression of the LDHA gene, thereby substantially reducing or eliminating the production of LDH, thereby reducing urinary oxalate and increasing serum glycolate.
  • a guide RNA with an RNA-guided DNA binding agent such as the CRISPR/Cas system to substantially reduce or knockout expression of the LDHA gene, thereby substantially reducing or eliminating the production of LDH, thereby reducing urinary oxalate and increasing serum glycolate.
  • the substantial reduction or elimination of the production of LDH through alteration of the LDHA gene can be a long-term or permanent treatment for hyperoxaluria.
  • Embodiment 01 A method of inducing a double-stranded break (DSB) or single-stranded break (SSB) within the LDHA gene, comprising delivering a composition to a cell, wherein the composition comprises:
  • compositions or formulation of any of the foregoing embodiments for the preparation of a medicament for treating a human subject having hyperoxaluria. Also disclosed are any of the foregoing compositions or formulations for use in treating hyperoxaluria or for use in modifying (e.g., forming an indel in, or forming a frameshift or nonsense mutation in) a LDHA gene.
  • FIG. 1 shows off-target analysis of certain sgRNAs targeting LDHA.
  • FIG. 2 shows dose response curves of editing % of certain sgRNAs targeting LDHA in PHH.
  • FIG. 3 shows dose response curves of editing % of certain sgRNAs targeting LDHA in PCH.
  • FIG. 4 shows Western Blot analysis of LDHA-targeted modified sgRNAs (listed in Table 2) in PHH.
  • FIG. 5 shows urine oxalate levels after treatment with LNPs comprising a modified sgRNAs in vivo in AGT-deficient mice.
  • FIG. 6 shows urine oxalate levels after treatment with LNPs comprising a modified sgRNA in vivo in AGT-deficient mice in a 15-week study.
  • FIG. 7 shows Western Blot analysis after treatment with LNPs comprising a modified sgRNA in vivo in AGT-deficient mice in a 15-week study.
  • FIG. 8 shows immunohistochemical staining of LDHA protein in vivo in livers of AGT-deficient mice.
  • FIG. 9 shows the correlation between the editing and protein levels depicted in Table 19.
  • FIG. 10 labels the 10 conserved region YA sites in an exemplary sgRNA sequence from 1 to 10 (SEQ ID NO: 2082).
  • the numbers 25, 45, 50, 56, 64, 67, and 83 indicate the position of the pyrimidine of YA sites 1, 5, 6, 7, 8, 9, and 10 in an sgRNA with a guide region indicated as (N)x, e.g., wherein x is optionally 20.
  • FIG. 11 shows an exemplary sgRNA (SEQ ID NO: 401; not all modifications are shown) in a possible secondary structure with labels designating individual nucleotides of the conserved region of the sgRNA, including the lower stem, bulge, upper stem, nexus (the nucleotides of which can be referred to as N1 through N18, respectively, in the 5′ to 3′ direction), hairpin 1, and hairpin 2 regions.
  • a nucleotide between hairpin 1 and hairpin 2 is labeled n.
  • a guide region may be present on an sgRNA and is indicated in this figure as “(N)x” preceding the conserved region of the sgRNA.
  • FIGS. 12A-12C show dose response curves of percent editing of certain sgRNAs targeting LDHA in primary cynomolgus hepatocytes.
  • FIGS. 13A-13B show dose response curves of relative reduction in LDHA expression after lipofection treatment comprising certain sgRNAs in primary human and cynomolgus hepatocytes.
  • FIGS. 14A-14C show dose-dependent urine oxalate levels, percent editing, and correlation between the urine oxalate levels and percent editing, respectively, after treatment with LNPs comprising a certain sgRNA of AGT-deficient mice.
  • FIGS. 15A-15B show LDHA activity in liver and muscle samples after treatment with LNPs comprising a certain sgRNA of AGT-deficient mice in the 15-week durability study as described in Example 4.
  • FIGS. 16A-16B show pyruvate levels in liver and plasma samples, after treatment with LNPs comprising a certain sgRNA of AGT-deficient mice in the 15-week durability study as described in Example 4.
  • FIG. 17 shows the average plasma lactate clearance function in mice that had undergone either 5/6 nephrectomy or sham surgeries after treatment with LNPs comprising a certain sgRNA.
  • Polynucleotide and “nucleic acid” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof.
  • a nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.
  • Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2′ methoxy or 2′ halide substitutions.
  • Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N 4 -methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidines, 4-amino-pyrim
  • Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Pat. No. 5,585,481).
  • a nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2′ methoxy linkages, or polymers containing both conventional bases and one or more base analogs).
  • Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004 , Biochemistry 43(42):13233-41).
  • LNA locked nucleic acid
  • RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.
  • RNA refers to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA).
  • the crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA).
  • sgRNA single guide RNA
  • dgRNA dual guide RNA
  • gRNA dual guide RNA
  • the trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences.
  • a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent.
  • a “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.”
  • a guide sequence can be 20 base pairs in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs/orthologs.
  • the guide sequence comprises at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:1-84.
  • the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence.
  • the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:1-84.
  • the guide sequence and the target region may be 100% complementary or identical.
  • the guide sequence and the target region may contain at least one mismatch.
  • the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs.
  • the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.
  • Target sequences for RNA-guided DNA binding agents include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for an RNA-guided DNA binding agent is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.
  • a “YA site” refers to a 5′-pyrimidine-adenine-3′ dinucleotide.
  • a “conserved region YA site” is present in the conserved region of an sgRNA.
  • a “guide region YA site” is present in the guide region of an sgRNA.
  • An unmodified YA site in an sgRNA may be susceptible to cleavage by RNase-A like endonucleases, e.g., RNase A.
  • an sgRNA comprises about 10 YA sites in its conserved region.
  • an sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites in its conserved region. Exemplary conserved region YA sites are indicated in FIG.
  • an sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the YA sites indicated in FIG. 10 .
  • an sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites at the following positions or a subset thereof: LS5-LS6; US3-US4; US9-US10; US12-B3; LS7-LS8; LS12-N1; N6-N7; N14-N15; N17-N18; and H2-2 to H2-3.
  • a YA site comprises a modification, meaning that at least one nucleotide of the YA site is modified.
  • the pyrimidine (also called the pyrimidine position) of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the pyrimidine).
  • the adenine (also called the adenine position) of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the adenine).
  • the pyrimidine position and the adenine position of the YA site comprise modifications.
  • RNA-guided DNA binding agent means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA.
  • RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof (“dCas DNA binding agents”).
  • Cas nuclease also called “Cas protein” as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents.
  • Cas cleavases/nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases.
  • a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-guided DNA binding activity, such as a Cas9 nuclease or a Cpf1 nuclease.
  • Class 2 Cas nucleases include Class 2 Cas cleavases and Class 2 Cas nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase/nickase activity is inactivated.
  • Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain.
  • Cas9 e.g., N497A, R661A, Q695A, Q926A variants
  • Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3.
  • “Cas9” encompasses Spy Cas9, the variants of Cas9 listed herein, and equivalents thereof. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).
  • ribonucleoprotein or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9).
  • a Cas nuclease e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9).
  • the guide RNA guides the RNA-guided DNA binding agent such as Cas9 to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking.
  • a first sequence is considered to “comprise a sequence with at least X % identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence.
  • the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence.
  • RNA and DNA generally the exchange of uridine for thymidine or vice versa
  • nucleoside analogs such as modified uridines
  • adenosine for all of thymidine, uridine, or modified uridine another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement.
  • sequence 5′-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5′-CAU).
  • exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art.
  • Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.
  • mRNA is used herein to refer to a polynucleotide that is RNA or modified RNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs).
  • mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2′-methoxy ribose residues.
  • the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2′-methoxy ribose residues, or a combination thereof.
  • “indels” refer to insertion/deletion mutations consisting of a number of nucleotides that are either inserted or deleted at the site of double-stranded breaks (DSBs) in a target nucleic acid.
  • knockdown refers to a decrease in expression of a particular gene product (e.g., protein, mRNA, or both). Knockdown of a protein can be measured by detecting total cellular amount of the protein from a tissue or cell population of interest. Methods for measuring knockdown of mRNA are known and include sequencing of mRNA isolated from a tissue or cell population of interest. In some embodiments, “knockdown” may refer to some loss of expression of a particular gene product, for example a decrease in the amount of mRNA transcribed or a decrease in the amount of protein expressed by a population of cells (including in vivo populations such as those found in tissues).
  • knockout refers to a loss of expression of a particular protein in a cell. Knockout can be measured either by detecting total cellular amount of a protein in a cell, a tissue or a population of cells. In some embodiments, the methods of the disclosure “knockout” LDHA in one or more cells (e.g., in a population of cells including in vivo populations such as those found in tissues). In some embodiments, a knockout is not the formation of mutant LDHA protein, for example, created by indels, but rather the complete loss of expression of LDH protein in a cell.
  • LDH refers to lactate dehydrogenase, which is the gene product of a LDHA gene. The human wild-type LDHA sequence is available at NCBI Gene ID: 3939; Ensembl ENSG00000134333.
  • “Hyperoxaluria” is a condition characterized by excess oxalate in the urine.
  • Exemplary types of hyperoxaluria include primary hyperoxaluria (including types 1 (PH1), 2 (PH2), and 3 (PH3)), oxalosis, enteric hyperoxaluria, and hyperoxaluria related to eating high-oxalate foods.
  • Hyperoxaluria may be idiopathic. High oxalate levels lead to calcium oxalate stone formation and renal parenchyma damage, which results in progressive deterioration of renal function and, eventually, end-stage renal disease. Thus, hyperoxaluria may result in excessive oxalate production and deposition of calcium oxalate crystals in the kidneys and urinary tract.
  • Renal damage from oxalate is caused by a combination of tubular toxicity, calcium oxalate deposition in the kidneys, and urinary obstruction by calcium oxalate stones.
  • Compromised kidney function exacerbates the disease as the excess oxalate can no longer be effectively excreted, resulting in subsequent accumulation and crystallization of oxalate in bones, eyes, skin, and heart, and other organs leading to severe illness and death. Kidney failure and end stage renal disease may occur. There are no approved pharmaceutical therapies for hyperoxaluria.
  • PH1 Primary Hyperoxaluria Type 1 (PH1) is an autosomal recessive disorder due to mutation of the AGXT gene, which encodes the liver peroxisomal alanine-glyoxylate aminotransferase (AGT) enzyme. AGT metabolizes glyoxylate to glycine. The lack of AGT activity, or its mistargeting to mitochondria, allows the oxidation of glyoxylate to oxalate, which can only be excreted in the urine.
  • AGTT liver peroxisomal alanine-glyoxylate aminotransferase
  • LDH lactate dehydrogenase
  • LDH activity should inhibit oxalate production resulting in decreased urinary oxalate levels while causing an accumulation of glyoxylate that may be converted to glycolate by glyoxylate reductase/hydroxypyruvate reductase (GRHPR).
  • GSHPR glyoxylate reductase/hydroxypyruvate reductase
  • glycolate is soluble and readily excreted in the urine.
  • methods for inhibiting LDH activity are provided, wherein once inhibited, oxalate production is inhibited and glycolate production is increased.
  • Oxalate an oxidation product of glyoxylate, can only be excreted in the urine.
  • High levels of oxalate in the urine (“hyperoxaluria”) is a symptom of hyperoxaluria.
  • increased oxalate in the urine is a symptom of hyperoxaluria.
  • Oxalate can combine with calcium to form calcium oxalate, which is the main component of kidney and bladder stones.
  • Deposits of calcium oxalate in the kidneys and other tissues can lead to blood in the urine (hematuria), urinary tract infections, kidney damage, end stage renal disease and others. Over time, oxalate levels in the blood may rise and calcium oxalate may be deposited in other organs throughout the body (oxalosis or systemic oxalosis).
  • a “target sequence” refers to a sequence of nucleic acid in a target gene that has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence.
  • treatment refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease.
  • treatment of hyperoxaluria may comprise alleviating symptoms of hyperoxaluria.
  • oxalate or “oxalate levels within a therapeutic range,” as used herein, means a greater than 30% reduction of urinary oxalate excretion as compared to baseline. See, Leumann and Hoppe (1999) Nephrol Dial Transplant 14:2556-2558 at 2557, second column. For example, achieving oxalate levels within a therapeutic range means reducing urinary oxalate greater than 30% from baseline.
  • a “normal oxalate level” or a “normal oxalate range” is between about 80 to about 122 ⁇ g oxalate/mg creatinine. See, Li et al.
  • a therapeutically relevant reduction of oxalate achieves levels of less than or within 200%, 150%, 125%, 120%, 115%, 110%, 105%, or 100% of normal.
  • compositions comprising Guide RNA (gRNAs)
  • compositions useful for inducing a double-stranded break (DSB) within the LDHA gene e.g., using a guide RNA with an RNA-guided DNA binding agent (e.g., a CRISPR/Cas system).
  • the compositions may be administered to subjects having or suspected of having hyperoxaluria.
  • the compositions may be administered to subjects having increased urinary oxalate output or decreased serum glycolate output.
  • Guide sequences targeting the LDHA gene are shown in Table 1 at SEQ ID NOs:1-84.
  • Each of the guide sequences shown in Table 1 at SEQ ID NOs:1-84 and 100-192 may further comprise additional nucleotides to form a crRNA, e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3′ end: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 200) in 5′ to 3′ orientation.
  • the above guide sequences may further comprise additional nucleotides to form a sgRNA, e.g., with the following exemplary nucleotide sequence following the 3′ end of the guide sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUU (SEQ ID NO: 201) or GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 203, which is SEQ ID NO: 201 without the four terminal U's) in 5′ to 3′ orientation.
  • the four terminal U's of SEQ ID NO: 201 are not present. In some embodiments, only 1, 2, or 3 of the four terminal U's of SEQ ID NO: 201 are present.
  • LDHA short-single guide RNAs comprising a guide sequence as described herein and a “conserved portion of an sgRNA” comprising a hairpin region, wherein the hairpin region lacks at least 5-10 nucleotides or 6-10 nucleotides.
  • a hairpin region of the LDHA short-single guide RNAs lacks 5-10 nucleotides with reference to the conserved portion of an sgRNA, e.g. nucleotides H1-1 to H2-15 in Table 2B.
  • a hairpin 1 region of the LDHA short-single guide RNAs lacks 5-10 nucleotides with reference to the conserved portion of an sgRNA, e.g. nucleotides H1-1 to H1-12 in Table 2B.
  • sgRNA S. pyogenes Cas9
  • Table 2A shows a “conserved region” of a S. pyogenes Cas9 (“spyCas9” (also referred to as “spCas9”)) sgRNA.
  • the first row shows the numbering of the nucleotides
  • the second row shows the sequence (SEQ ID NO: 700); and the third row shows “domains.”
  • Briner A E et al., Molecular Cell 56:333-339 (2014) describes functional domains of sgRNAs, referred to herein as “domains”, including the “spacer” domain responsible for targeting, the “lower stem”, the “bulge”, “upper stem” (which may include a tetraloop), the “nexus”, and the “hairpin 1” and “hairpin 2” domains. See, Briner et al. at page 334, FIG. 1A .
  • Table 2B provides a schematic of the domains of an sgRNA as used herein.
  • the “n” between regions represents a variable number of nucleotides, for example, from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, n equals 0. In some embodiments, n equals 1.
  • the LDHA sgRNA is from S. pyogenes Cas9 (“spyCas9”) or a spyCas9 equivalent. In some embodiments, the sgRNA is not from S. pyogenes (“non-spyCas9”). In some embodiments, the 5-10 nucleotides or 6-10 nucleotides are consecutive.
  • an LDHA short-sgRNA lacks at least nucleotides 54-58 (AAAAA) of the conserved portion of a S. pyogenes Cas9 (“spyCas9”) sgRNA, as shown in Table 2A.
  • an LDHA short-sgRNA is a non-spyCas9 sgRNA that lacks at least nucleotides corresponding to nucleotides 54-58 (AAAAA) of the conserved portion of a spyCas9 as determined, for example, by pairwise or structural alignment.
  • the non-spyCas9 sgRNA is Staphylococcus aureus Cas9 (“saCas9”) sgRNA.
  • an LDHA short-sgRNA lacks at least nucleotides 54-61 (AAAAAGUG) of the conserved portion of a spyCas9 sgRNA. In some embodiments, an LDHA short-sgRNA lacks at least nucleotides 53-60 (GAAAAAGU) of the conserved portion of a spyCas9 sgRNA.
  • an LDHA short-sgRNA lacks 4, 5, 6, 7, or 8 nucleotides of nucleotides 53-60 (GAAAAAGU) or nucleotides 54-61 (AAAAAGUG) of the conserved portion of a spyCas9 sgRNA, or the corresponding nucleotides of the conserved portion of a non-spyCas9 sgRNA as determined, for example, by pairwise or structural alignment.
  • the sgRNA comprises any one of the guide sequences of SEQ ID NOs: 1-146 and additional nucleotides to form a crRNA, e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3′ end: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GGCACCGAGUCGGUGC (SEQ ID NO: 202) in 5′ to 3′ orientation.
  • SEQ ID NO: 202 lacks 8 nucleotides with reference to a wild-type guide RNA conserved sequence:
  • the invention provides a composition comprising one or more guide RNA (gRNA) comprising guide sequences that direct an RNA-guided DNA binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in LDHA.
  • gRNA guide RNA
  • the gRNA may comprise a crRNA comprising a guide sequence shown in Table 1.
  • the gRNA may comprise a crRNA comprising 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1.
  • the gRNA comprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1.
  • the gRNA comprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guide sequence shown in Table 1.
  • the gRNA may further comprise a trRNA.
  • the crRNA and trRNA may be associated as a single RNA (sgRNA) or may be on separate RNAs (dgRNA).
  • sgRNA single RNA
  • dgRNA separate RNAs
  • the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.
  • the guide RNA may comprise two RNA molecules as a “dual guide RNA” or “dgRNA”.
  • the dgRNA comprises a first RNA molecule comprising a crRNA comprising, e.g., a guide sequence shown in Table 1, and a second RNA molecule comprising a trRNA.
  • the first and second RNA molecules may not be covalently linked but may form an RNA duplex via the base pairing between portions of the crRNA and the trRNA.
  • the guide RNA may comprise a single RNA molecule as a “single guide RNA” or “sgRNA”.
  • the sgRNA may comprise a crRNA (or a portion thereof) comprising a guide sequence shown in Table 1 covalently linked to a trRNA.
  • the sgRNA may comprise 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1.
  • the crRNA and the trRNA are covalently linked via a linker.
  • the sgRNA forms a stem-loop structure via the base pairing between portions of the crRNA and the trRNA.
  • the crRNA and the trRNA are covalently linked via one or more bonds that are not a phosphodiester bond.
  • the trRNA may comprise all or a portion of a trRNA sequence derived from a naturally-occurring CRISPR/Cas system.
  • the trRNA comprises a truncated or modified wild type trRNA.
  • the length of the trRNA depends on the CRISPR/Cas system used.
  • the trRNA comprises or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides.
  • the trRNA may comprise certain secondary structures, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.
  • the invention provides a composition comprising one or more guide RNAs comprising a guide sequence of any one of SEQ ID NOs:1-84.
  • the invention provides a composition comprising one or more sgRNAs comprising any one of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • the invention provides a composition comprising a gRNA that comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs:1-84.
  • the composition comprises at least one, e.g., at least two gRNA's comprising guide sequences selected from any two or more of the guide sequences of SEQ ID NOs:1-84.
  • the composition comprises at least two gRNA's that each comprise a guide sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs:1-84.
  • the guide RNA compositions of the present invention are designed to recognize (e.g., hybridize to) a target sequence in the LDHA gene.
  • the LDHA target sequence may be recognized and cleaved by a provided Cas cleavase comprising a guide RNA.
  • an RNA-guided DNA binding agent such as a Cas cleavase
  • the selection of the one or more guide RNAs is determined based on target sequences within the LDHA gene.
  • a gRNA complementary or having complementarity to a target sequence within LDHA is used to direct the RNA-guided DNA binding agent to a particular location in the LDHA gene.
  • gRNAs are designed to have guide sequences that are complementary or have complementarity to target sequences in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7 or exon 8 of LDHA.
  • the guide sequence is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a target sequence present in the human LDHA gene.
  • the target sequence may be complementary to the guide sequence of the guide RNA.
  • the degree of complementarity or identity between a guide sequence of a guide RNA and its corresponding target sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the target sequence and the guide sequence of the gRNA may be 100% complementary or identical.
  • the target sequence and the guide sequence of the gRNA may contain at least one mismatch.
  • the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, or 4 mismatches, where the total length of the guide sequence is 20.
  • the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches where the guide sequence is 20 nucleotides.
  • a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein.
  • an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease is provided, used, or administered.
  • the gRNA is chemically modified.
  • a gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues.
  • a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.”
  • Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the rib
  • modified gRNAs and/or mRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications.
  • a modified residue can have a modified sugar and a modified nucleobase.
  • every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group.
  • all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups.
  • modified gRNAs comprise at least one modified residue at or near the 5′ end of the RNA.
  • modified gRNAs comprise at least one modified residue at or near the 3′ end of the RNA.
  • the gRNA comprises one, two, three or more modified residues.
  • at least 5% e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
  • modified nucleosides or nucleotides are modified nucleosides or nucleotides.
  • Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum.
  • nucleases can hydrolyze nucleic acid phosphodiester bonds.
  • the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases.
  • the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo.
  • the term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
  • the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent.
  • the modified residue e.g., modified residue present in a modified nucleic acid
  • the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
  • modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the nonbridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).
  • the backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • a bridging oxygen i.e., the oxygen that links the phosphate to the nucleoside
  • nitrogen bridged phosphoroamidates
  • sulfur bridged phosphorothioates
  • carbon bridged methylenephosphonates
  • the phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications.
  • the charged phosphate group can be replaced by a neutral moiety.
  • moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications.
  • the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
  • the modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification.
  • the 2′ hydroxyl group (OH) can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents.
  • modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion.
  • Examples of 2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH 2 CH 2 O) n CH 2 CH 2 OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20).
  • R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar
  • PEG polyethylene
  • the 2′ hydroxyl group modification can be 2′-O-Me. In some embodiments, the 2′ hydroxyl group modification can be a 2′-fluoro modification, which replaces the 2′ hydroxyl group with a fluoride.
  • the 2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH 2 ) n -amino, (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino
  • the 2′ hydroxyl group modification can include “unlocked” nucleic acids (UNA) in which the ribose ring lacks the C2′—C3′ bond.
  • the 2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH 2 CH 2 OCH 3 , e.g., a PEG derivative).
  • “Deoxy” 2′ modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH 2 CH 2 NH) n CH 2 CH 2 — amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycl
  • the sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar.
  • the modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms.
  • the modified nucleic acids can also include one or more sugars that are in the L form, e.g. L-nucleosides.
  • the modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase.
  • a modified base also called a nucleobase.
  • nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids.
  • the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog.
  • the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.
  • each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA.
  • one or more residues at one or both ends of the sgRNA may be chemically modified, and/or internal nucleosides may be modified, and/or the entire sgRNA may be chemically modified.
  • Certain embodiments comprise a 5′ end modification.
  • Certain embodiments comprise a 3′ end modification.
  • the guide RNAs disclosed herein comprise one of the modification patterns disclosed in WO2018/107028 A1, filed Dec. 8, 2017, titled “Chemically Modified Guide RNAs,” the contents of which are hereby incorporated by reference in their entirety.
  • the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in US20170114334, the contents of which are hereby incorporated by reference in their entirety.
  • the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2017/136794, the contents of which are hereby incorporated by reference in their entirety.
  • a modification at a YA site can be a modification of the internucleoside linkage, a modification of the base (pyrimidine or adenine), e.g. by chemical modification, substitution, or otherwise, and/or a modification of the sugar (e.g. at the 2′ position, such as 2′-O-alkyl, 2′-F, 2′-moe, 2′-F arabinose, 2′-H (deoxyribose), and the like).
  • a “YA modification” is any modification that alters the structure of the dinucleotide motif to reduce RNA endonuclease activity, e.g., by interfering with recognition or cleavage of a YA site by an RNase and/or by stabilizing an RNA structure (e.g., secondary structure) that decreases accessibility of a cleavage site to an RNase.
  • an RNA structure e.g., secondary structure
  • Peacock et al., Belhke, Ku, and Ghidini provide exemplary modifications suitable as YA modifications. Modifications known to those of skill in the art to reduce endonucleolytic degradation are encompassed. Exemplary 2′ ribose modifications that affect the 2′ hydroxyl group involved in RNase cleavage are 2′-H and 2′-O-alkyl, including 2′-O-Me. Modifications such as bicyclic ribose analogs, UNA, and modified internucleoside linkages of the residues at the YA site can be YA modifications. Exemplary base modifications that can stabilize RNA structures are pseudouridine and 5-methylcytosine. In some embodiments, at least one nucleotide of the YA site is modified.
  • the pyrimidine (also called “pyrimidine position”) of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the pyrimidine, a modification of the pyrimidine base, and a modification of the ribose, e.g. at its 2′ position).
  • the adenine (also called “adenine position”) of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the pyrimidine, a modification of the pyrimidine base, and a modification of the ribose, e.g. at its 2′ position).
  • the pyrimidine and the adenine of the YA site comprise modifications.
  • the YA modification reduces RNA endonuclease activity.
  • an sgRNA comprises modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more YA sites.
  • the pyrimidine of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the pyrimidine).
  • the adenine of the YA site comprises a modification (which includes a modification altering the internucleoside linkage immediately 3′ of the sugar of the adenine).
  • the pyrimidine and the adenine of the YA site comprise modifications, such as sugar, base, or internucleoside linkage modifications.
  • the YA modifications can be any of the types of modifications set forth herein.
  • the YA modifications comprise one or more of phosphorothioate, 2′-OMe, or 2′-fluoro. In some embodiments, the YA modifications comprise pyrimidine modifications comprising one or more of phosphorothioate, 2′-OMe, or 2′-fluoro. In some embodiments, the YA modification comprises a bicyclic ribose analog (e.g., an LNA, BNA, or ENA) within an RNA duplex region that contains one or more YA sites.
  • a bicyclic ribose analog e.g., an LNA, BNA, or ENA
  • the YA modification comprises a bicyclic ribose analog (e.g., an LNA, BNA, or ENA) within an RNA duplex region that contains a YA site, wherein the YA modification is distal to the YA site.
  • a bicyclic ribose analog e.g., an LNA, BNA, or ENA
  • the sgRNA comprises a guide region YA site modification.
  • the guide region comprises 1, 2, 3, 4, 5, or more YA sites (“guide region YA sites”) that may comprise YA modifications.
  • one or more YA sites located at 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′ terminus (where “5-end”, etc., refers to position 5 to the 3′ end of the guide region, i.e., the most 3′ nucleotide in the guide region) comprise YA modifications.
  • two or more YA sites located at 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′ terminus comprise YA modifications.
  • three or more YA sites located at 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′ terminus comprise YA modifications.
  • four or more YA sites located at 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′ terminus comprise YA modifications.
  • YA sites located at 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5′ end of the 5′ terminus comprise YA modifications.
  • a modified guide region YA site comprises a YA modification.
  • a modified guide region YA site is within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3′ terminal nucleotide of the guide region.
  • a modified guide region YA site is within 10 nucleotides of the 3′ terminal nucleotide of the guide region and the guide region is 20 nucleotides long, then the modified nucleotide of the modified guide region YA site is located at any of positions 11-20.
  • a YA modification is located within a YA site 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides from the 3′ terminal nucleotide of the guide region.
  • a YA modification is located 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides from the 3′ terminal nucleotide of the guide region.
  • a modified guide region YA site is at or after nucleotide 4, 5, 6, 7, 8, 9, 10, or 11 from the 5′ end of the 5′ terminus.
  • a modified guide region YA site is other than a 5′ end modification.
  • an sgRNA can comprise a 5′ end modification as described herein and further comprise a modified guide region YA site.
  • an sgRNA can comprise an unmodified 5′ end and a modified guide region YA site.
  • an sgRNA can comprise a modified 5′ end and an unmodified guide region YA site.
  • a modified guide region YA site comprises a modification that at least one nucleotide located 5′ of the guide region YA site does not comprise.
  • nucleotides 1-3 comprise phosphorothioates
  • nucleotide 4 comprises only a 2′-OMe modification
  • nucleotide 5 is the pyrimidine of a YA site and comprises a phosphorothioate
  • the modified guide region YA site comprises a modification (phosphorothioate) that at least one nucleotide located 5′ of the guide region YA site (nucleotide 4) does not comprise.
  • nucleotides 1-3 comprise phosphorothioates
  • nucleotide 4 is the pyrimidine of a YA site and comprises a 2′-OMe
  • the modified guide region YA site comprises a modification (2′-OMe) that at least one nucleotide located 5′ of the guide region YA site (any of nucleotides 1-3) does not comprise. This condition is also always satisfied if an unmodified nucleotide is located 5′ of the modified guide region YA site.
  • the modified guide region YA sites comprise modifications as described for YA sites above.
  • the sgRNA comprises a conserved region YA site modification.
  • conserved region YA sites 1-10 are illustrated in FIG. 10 .
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved region YA sites comprise modifications.
  • conserved region YA sites 1, 8, or 1 and 8 comprise YA modifications. In some embodiments, conserved region YA sites 1, 2, 3, 4, and 10 comprise YA modifications. In some embodiments, YA sites 2, 3, 4, 8, and 10 comprise YA modifications. In some embodiments, conserved region YA sites 1, 2, 3, and 10 comprise YA modifications. In some embodiments, YA sites 2, 3, 8, and 10 comprise YA modifications. In some embodiments, YA sites 1, 2, 3, 4, 8, and 10 comprise YA modifications. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 additional conserved region YA sites comprise YA modifications.
  • 1, 2, 3, or 4 of conserved region YA sites 2, 3, 4, and 10 comprise YA modifications. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 additional conserved region YA sites comprise YA modifications.
  • the modified conserved region YA sites comprise modifications as described for YA sites above.
  • the sgRNA comprises any of the modification patterns shown above in Table 2, or below in Table 3, where N, if present, is any natural or non-natural nucleotide, and wherein the totality of the N's comprise an LDHA guide sequence as described herein in Table 1.
  • Table 3 does not depict the guide sequence portion of the sgRNA. The modifications remain as shown in Table 3 despite the substitution of N's for the nucleotides of a guide. That is, although the nucleotides of the guide replace the “N's”, the nucleotides are modified as shown in Table 3. When the guide sequence is appended to the 5′ end, the 5′ end (or 5′ terminus) of the guide sequence may be modified.
  • the modifications comprise 2′-O-Me and/or PS-bonds.
  • the 2′-O-Me and/or PS-bonds are at the first 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 nucleotides of the guide sequence at its 5′ end.
  • the modified sgRNA comprises the following sequence: mN*mN*mN NNGUUUUAGAmGmCmUmAmGmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU*mU (SEQ ID NO: 300), where “N” may be any natural or non-natural nucleotide, and wherein the totality of N's comprise an LDHA guide sequence as described in Table 1.
  • SEQ ID NO: 300 encompassed herein is SEQ ID NO: 300, where the N's are replaced with any of the guide sequences disclosed herein in Table 1 (SEQ ID NOs: 1-84).
  • mA mA
  • mC mU
  • mG mG
  • nucleotide sugar rings Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution.
  • 2′-fluoro (2′-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.
  • fA fC
  • fU fU
  • Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases.
  • PS Phosphorothioate
  • the modified oligonucleotides may also be referred to as S-oligos.
  • a “*” may be used to depict a PS modification.
  • the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3′) nucleotide with a PS bond.
  • mA* may be used to denote a nucleotide that has been substituted with 2′-O-Me and that is linked to the next (e.g., 3′) nucleotide with a PS bond.
  • Abasic nucleotides refer to those which lack nitrogenous bases.
  • the figure below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base:
  • Inverted bases refer to those with linkages that are inverted from the normal 5′ to 3′ linkage (i.e., either a 5′ to 5′ linkage or a 3′ to 3′ linkage). For example:
  • An abasic nucleotide can be attached with an inverted linkage.
  • an abasic nucleotide may be attached to the terminal 5′ nucleotide via a 5′ to 5′ linkage, or an abasic nucleotide may be attached to the terminal 3′ nucleotide via a 3′ to 3′ linkage.
  • An inverted abasic nucleotide at either the terminal 5′ or 3′ nucleotide may also be called an inverted abasic end cap.
  • one or more of the first three, four, or five nucleotides at the 5′ terminus, and one or more of the last three, four, or five nucleotides at the 3′ terminus are modified.
  • the modification is a 2′-O-Me, 2′-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and/or performance.
  • the first four nucleotides at the 5′ terminus, and the last four nucleotides at the 3′ terminus are linked with phosphorothioate (PS) bonds.
  • PS phosphorothioate
  • the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise an inverted abasic nucleotide.
  • the guide RNA comprises a modified sgRNA.
  • the sgRNA comprises the modification pattern shown in SEQ ID No: 201, 202, or 203, where N is any natural or non-natural nucleotide, and where the totality of the N's comprise a guide sequence that directs a nuclease to a target sequence in LDHA, e.g., as shown in Table 1.
  • the guide RNA comprises a sgRNA shown in any one of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • the guide RNA comprises a sgRNA comprising any one of the guide sequences of SEQ ID No: 1-84 and 100-192 and the nucleotides of SEQ ID No: 201, 202, or 203, wherein the nucleotides of SEQ ID No: 201, 202, or 203 are on the 3′ end of the guide sequence, and wherein the sgRNA may be modified as shown in Table 3 or SEQ ID NO: 300.
  • a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein.
  • an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease is provided, used, or administered.
  • the ORF encoding an RNA-guided DNA nuclease is a “modified RNA-guided DNA binding agent ORF” or simply a “modified ORF,” which is used as shorthand to indicate that the ORF is modified.
  • the modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions.
  • the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl.
  • the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl.
  • the modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof.
  • the modified uridine is 5-methoxyuridine.
  • the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.
  • an mRNA disclosed herein comprises a 5′ cap, such as a Cap0, Cap1, or Cap2.
  • a 5′ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g. with respect to ARCA) linked through a 5′-triphosphate to the 5′ position of the first nucleotide of the 5′-to-3′ chain of the mRNA, i.e., the first cap-proximal nucleotide.
  • the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-hydroxyl.
  • the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2′-methoxy and a 2′-hydroxyl, respectively.
  • the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115.
  • Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2.
  • Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as “non-self” by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon.
  • components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA.
  • a cap can be included co-transcriptionally.
  • ARCA anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045
  • ARCA is a cap analog comprising a 7-methylguanine 3′-methoxy-5′-triphosphate linked to the 5′ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation.
  • ARCA results in a Cap0 cap in which the 2′ position of the first cap-proximal nucleotide is hydroxyl.
  • CleanCapTM AG (m7G(5′)ppp(5′)(2′OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCapTM GG (m7G(5′)ppp(5′)(2′OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally.
  • 3′-O-methylated versions of CleanCapTM AG and CleanCapTM GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively.
  • the CleanCapTM AG structure is shown below.
  • a cap can be added to an RNA post-transcriptionally.
  • Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit.
  • it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J Biol. Chem. 269, 24472-24479.
  • the mRNA further comprises a poly-adenylated (poly-A) tail.
  • the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines.
  • the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides.
  • a composition comprising one or more gRNAs comprising one or more guide sequences from Table 1 or one or more sgRNAs from Table 2 and an RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9.
  • the RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity.
  • the RNA-guided DNA-binding agent comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, S.
  • Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof.
  • the Cas nuclease may be from a Type-IIA, Type-IIB, or Type-IIC system.
  • Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis , Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides
  • the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes . In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus thermophilus . In some embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis . In some embodiments, the Cas nuclease is the Cas9 nuclease is from Staphylococcus aureus . In some embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella novicida .
  • the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceae bacterium ND2006.
  • the Cas nuclease is the Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens , or Porphyromonas macacae .
  • the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae.
  • the gRNA together with an RNA-guided DNA binding agent is called a ribonucleoprotein complex (RNP).
  • the RNA-guided DNA binding agent is a Cas nuclease.
  • the gRNA together with a Cas nuclease is called a Cas RNP.
  • the RNP comprises Type-I, Type-II, or Type-III components.
  • the Cas nuclease is the Cas9 protein from the Type-II CRISPR/Cas system.
  • the gRNA together with Cas9 is called a Cas9 RNP.
  • Wild type Cas9 has two nuclease domains: RuvC and HNH.
  • the RuvC domain cleaves the non-target DNA strand
  • the HNH domain cleaves the target strand of DNA.
  • the Cas9 protein comprises more than one RuvC domain and/or more than one HNH domain.
  • the Cas9 protein is a wild type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in target DNA.
  • chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein.
  • a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1.
  • a Cas nuclease may be a modified nuclease.
  • the Cas nuclease may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.
  • the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.”
  • the RNA-guided DNA-binding agent comprises a Cas nickase.
  • a nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix.
  • a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations.
  • a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain.
  • the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain.
  • the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.
  • a nickase is used having a RuvC domain with reduced activity.
  • a nickase is used having an inactive RuvC domain.
  • a nickase is used having an HNH domain with reduced activity.
  • a nickase is used having an inactive HNH domain.
  • a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity.
  • a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain.
  • Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell October 22:163(3): 759-771.
  • the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain.
  • Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2 (CPF1_FRATN)).
  • an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively.
  • the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking).
  • double nicking may improve specificity and reduce off-target effects.
  • a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA.
  • a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.
  • the RNA-guided DNA-binding agent lacks cleavase and nickase activity.
  • the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide.
  • a dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity.
  • the dCas polypeptide is a dCas9 polypeptide.
  • the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014/0186958 A1; US 2015/0166980 A1.
  • the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).
  • the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell.
  • the heterologous functional domain may be a nuclear localization signal (NLS).
  • the RNA-guided DNA-binding agent may be fused with 1-10 NLS(s).
  • the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s).
  • the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence.
  • the RNA-guided DNA-binding agent may be fused with more than one NLS. In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus.
  • the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS. In some embodiments, the NLS may be a monopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 600) or PKKKRRV (SEQ ID NO: 601).
  • the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 602).
  • a single PKKKRKV (SEQ ID NO: 600) NLS may be linked at the C-terminus of the RNA-guided DNA-binding agent.
  • One or more linkers are optionally included at the fusion site.
  • the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation.
  • the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases.
  • the heterologous functional domain may comprise a PEST sequence.
  • the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain.
  • the ubiquitin may be a ubiquitin-like protein (UBL).
  • Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rubl in S. cerevisiae ), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBLS).
  • SUMO small ubiquitin-like modifier
  • URP ubiquitin cross-reactive protein
  • ISG15 interferon-stimulated gene-15
  • UDM1 ubiquitin-related modifier-1
  • NEDD8 neuronal-precursor-cell-
  • the heterologous functional domain may be a marker domain.
  • marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences.
  • the marker domain may be a fluorescent protein.
  • suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g.,
  • the marker domain may be a purification tag and/or an epitope tag.
  • Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6 ⁇ His, 8 ⁇ His, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • MBP maltose binding protein
  • TRX thioredoxin
  • poly(NANP) tandem affinity purification
  • TAP tandem affinity pur
  • Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-glucuronidase
  • luciferase or fluorescent proteins.
  • the heterologous functional domain may target the RNA-guided DNA-binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to mitochondria.
  • the heterologous functional domain may be an effector domain.
  • the effector domain may modify or affect the target sequence.
  • the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • the heterologous functional domain is a nuclease, such as a FokI nuclease.
  • the heterologous functional domain is a transcriptional activator or repressor.
  • a transcriptional activator or repressor See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol.
  • RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA.
  • the efficacy of a gRNA is determined when delivered or expressed together with other components forming an RNP.
  • the gRNA is expressed together with an RNA-guided DNA binding agent, such as a Cas protein, e.g. Cas9.
  • the gRNA is delivered to or expressed in a cell line that already stably expresses an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase.
  • the gRNA is delivered to a cell as part of an RNP.
  • the gRNA is delivered to a cell along with a mRNA encoding an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase.
  • a mRNA encoding an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase.
  • RNA-guided DNA nuclease and a guide RNA disclosed herein can lead to double-stranded breaks in the DNA which can produce errors in the form of insertion/deletion (indel) mutations upon repair by cellular machinery.
  • Indel insertion/deletion
  • Many mutations due to indels alter the reading frame or introduce premature stop codons and, therefore, produce a non-functional protein.
  • the efficacy of particular gRNAs is determined based on in vitro models.
  • the in vitro model is HEK293 cells stably expressing Cas9 (HEK293 Cas9).
  • the in vitro model is HUH7 human hepatocarcinoma cells.
  • the in vitro model is HepG2 cells.
  • the in vitro model is primary human hepatocytes.
  • the in vitro model is primary cynomolgus hepatocytes. With respect to using primary human hepatocytes, commercially available primary human hepatocytes can be used to provide greater consistency between experiments.
  • the number of off-target sites at which a deletion or insertion occurs in an in vitro model is determined, e.g., by analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA and the guide RNA.
  • such a determination comprises analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide. Exemplary procedures for such determinations are provided in the working examples below.
  • the efficacy of particular gRNAs is determined across multiple in vitro cell models for a gRNA selection process.
  • a cell line comparison of data with selected gRNAs is performed.
  • cross screening in multiple cell models is performed.
  • the efficacy of particular gRNAs is determined based on in vivo models.
  • the in vivo model is a rodent model.
  • the rodent model is a mouse which expresses a LDHA gene.
  • the rodent model is a mouse which expresses a human LDHA gene.
  • the in vivo model is a non-human primate, for example cynomolgus monkey.
  • the efficacy of a guide RNA is measured by percent editing of LDHA.
  • the percent editing of LDHA is compared to the percent editing necessary to achieve knockdown of LDHA protein, e.g., from whole cell lysates in the case of an in vitro model or in tissue in the case of an in vivo model.
  • the efficacy of a guide RNA is measured by the number and/or frequency of indels at off-target sequences within the genome of the target cell type.
  • efficacious guide RNAs are provided which produce indels at off target sites at very low frequencies (e.g., ⁇ 5%) in a cell population and/or relative to the frequency of indel creation at the target site.
  • the disclosure provides for guide RNAs which do not exhibit off-target indel formation in the target cell type (e.g., a hepatocyte), or which produce a frequency of off-target indel formation of ⁇ 5% in a cell population and/or relative to the frequency of indel creation at the target site.
  • the disclosure provides guide RNAs which do not exhibit any off target indel formation in the target cell type (e.g., hepatocyte).
  • guide RNAs are provided which produce indels at less than 5 off-target sites, e.g., as evaluated by one or more methods described herein.
  • guide RNAs are provided which produce indels at less than or equal to 4, 3, 2, or 1 off-target site(s) e.g., as evaluated by one or more methods described herein.
  • the off-target site(s) does not occur in a protein coding region in the target cell (e.g., hepatocyte) genome.
  • detecting gene editing events such as the formation of insertion/deletion (“indel”) mutations and homology directed repair (HDR) events in target DNA utilize linear amplification with a tagged primer and isolating the tagged amplification products (herein after referred to as “LAM-PCR,” or “Linear Amplification (LA)” method).
  • Indel insertion/deletion
  • HDR homology directed repair
  • the efficacy of a guide RNA is measured by mearing levels of glycolate and/or levels of oxalate in a sample such as a body fluid, e.g., serum, plasma, blood, or urine.
  • a body fluid e.g., serum, plasma, blood, or urine.
  • the efficacy of a guide RNA is measured by mearing levels of glycolate in the serum or plasma and/or levels of oxalate in the urine.
  • An increase in the levels of glycolate in the serum or plasma and/or a decrease in the level of oxalate in the urine is indicative of an effective guide RNA.
  • urinary oxalate is reduced below 0.7 mmol/24 hrs/1.73 m 2 .
  • levels of glycolate and oxalate are measured using an enzyme-linked immunosorbent assay (ELISA) assay with cell culture media or serum or plasma.
  • ELISA enzyme-linked immunosorbent assay
  • levels of glycolate and oxalate are measured in the same in vitro or in vivo systems or models used to measure editing.
  • levels of glycolate and oxalate are measured in cells, e.g., primary human hepatocytes.
  • levels of glycolate and oxalate are measured in HUH7 cells.
  • levels of glycolate and oxalate are measured in HepG2 cells.
  • the gRNAs and associated methods and compositions disclosed herein are useful in inducing a double-stranded break (DSB) within the LDHA gene and reducing the expression of the LDHA gene.
  • the gRNAs and associated methods and compositions disclosed herein are useful in treating and preventing hyperoxaluria and preventing symptoms of hyperoxaluria.
  • the gRNAs disclosed herein are useful in treating and preventing calcium oxalate production, calcium oxalate deposition in organs, primary hyperoxaluria (including PH1, PH2, and PH3), oxalosis, including systemic oxalosis, and hematuria.
  • the gRNAs disclosed herein are useful in delaying or ameliorating the need for kidney or liver transplant. In some embodiments, the gRNAs disclosed herein are useful in preventing end stage renal disease (ESRD).
  • Administration of the gRNAs disclosed herein will increase serum or plasma glycolate and decrease oxalate production or accumulation so that less oxalate is excreted in the urine. Therefore, in one aspect, effectiveness of treatment/prevention can be assessed by measuring serum or plasma glycolate, wherein an increase in glycolate levels indicates effectiveness. In some embodiments, effectiveness of treatment/prevention can be assessed by measuring oxalate in a sample, such as urinary oxalate, wherein a decrease in urinary oxalate indicates effectiveness.
  • gRNAs and compositions disclosed herein are useful for reducing levels of oxalate such that a subject no longer exhibits levels of urinary oxalate associated with clinical hyperoxaluria.
  • administration of the gRNAs and compositions disclosed herein reduces a subject's urinary oxalate to less than about 45 or 40 mg in a 24-hour period. In some embodiments, administration of the gRNAs and compositions disclosed herein reduces a subject's urinary oxalate to less than about 35, less than about 30, less than about 25, less than about 20, less than about 15, or less than about 10 mg in a 24-hour period.
  • any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein is for use in preparing a medicament for treating or preventing a disease or disorder in a subject.
  • treatment and/or prevention is accomplished with a single dose, e.g., one-time treatment, of medicament/composition.
  • the disease or disorder is hyperoxaluria.
  • the invention comprises a method of treating or preventing a disease or disorder in subject comprising administering any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein.
  • the disease or disorder is hyperoxaluria.
  • the gRNAs, compositions, or pharmaceutical formulations described herein are administered as a single dose, e.g., at one time.
  • the single dose achieves durable treatment and/or prevention.
  • the method achieves durable treatment and/or prevention.
  • Durable treatment and/or prevention includes treatment and/or prevention that extends at least i) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, or 36 months; or iii) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • a single dose of the gRNAs, compositions, or pharmaceutical formulations described herein is sufficient to treat and/or prevent any of the indications described herein for the duration of the subject's life.
  • the invention comprises a method or use of modifying (e.g., creating a double strand break) a target DNA comprising, administering or delivering any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein.
  • the target DNA is the LDHA gene.
  • the target DNA is in an exon of the LDHA gene.
  • the target DNA is in exon 1, 2, 3, 4, 5, 6, 7, or 8 of the LDHA gene.
  • the invention comprises a method or use for modulation of a target gene comprising, administering or delivering any one or more of the gRNAs, compositions, or pharmaceutical formulations described herein.
  • the modulation is editing of the LDHA target gene.
  • the modulation is a change in expression of the protein encoded by the LDHA target gene.
  • the method or use results in gene editing. In some embodiments, the method or use results in a double-stranded break within the target LDHA gene. In some embodiments, the method or use results in formation of indel mutations during non-homologous end joining of the DSB. In some embodiments, the method or use results in an insertion or deletion of nucleotides in a target LDHA gene. In some embodiments, the insertion or deletion of nucleotides in a target LDHA gene leads to a frameshift mutation or premature stop codon that results in a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in a target LDHA gene leads to a knockdown or elimination of target gene expression. In some embodiments, the method or use comprises homology directed repair of a DSB.
  • the method or use results in LDHA gene modulation.
  • the LDHA gene modulation is a decrease in gene expression.
  • the method or use results in decreased expression of the protein encoded by the target gene.
  • a method of inducing a double-stranded break (DSB) within the LDHA gene comprising administering a composition comprising a guide RNA comprising any one or more guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs:1-84 and 100-192 are administered to induce a DSB in the LDHA gene.
  • the guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • a method of modifying the LDHA gene comprising administering a composition comprising a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081, are administered to modify the LDHA gene.
  • the guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • a method of treating or preventing hyperoxaluria comprising administering a composition comprising a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081 are administered to treat or prevent hyperoxaluria.
  • the guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • the hyperoxaluria is primary hyperoxaluria.
  • the primary hyperoxaluria is type 1 (PH1), type 2 (PH2), or type 3 (PH3).
  • the hyperoxaluria is idiopathic.
  • a method of decreasing or eliminating calcium oxalate production and/or deposition comprising administering a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • the guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • a method of treating or preventing primary hyperoxaluria comprising administering a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • the guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • a method of treating or preventing oxalosis, including systemic oxalosis comprising administering a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • the guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • a method of treating or preventing hematuria comprising administering a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs:1-84, or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081.
  • the guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs:1-84 and 100-192 or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081 are administered to reduce oxalate levels in the urine.
  • the gRNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • gRNAs comprising any one or more of the guide sequences of SEQ ID NOs:1-84 and 100-192 or any one or more of the sgRNAs of SEQ ID NOs: 1001, 1005, 1007, 1008, 1014, 1023, 1027, 1032, 1045, 1048, 1063, 1067, 1069, 1071, 1074, 1076, 1077, 1078, 1079, and 1081, or modified versions thereof as shown, e.g., in SEQ ID NOs: 2001, 2005, 2007, 2008, 2014, 2023, 2027, 2032, 2045, 2048, 2063, 2067, 2069, 2071, 2074, 2076, 2077, 2078, 2079, and 2081 are administered to increase serum glycolate in the serum or plasma.
  • the gRNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).
  • the gRNAs comprising the guide sequences of Table 1 together with an RNA-guided DNA nuclease such as a Cas nuclease induce DSBs, and non-homologous ending joining (NHEJ) during repair leads to a mutation in the LDHA gene.
  • NHEJ leads to a deletion or insertion of a nucleotide(s), which induces a frame shift or nonsense mutation in the LDHA gene.
  • administering the guide RNAs of the invention increases levels (e.g., serum or plasma levels) of glycolate in the subject, and therefore prevents oxalate accumulation.
  • levels e.g., serum or plasma levels
  • increasing serum glycolate results in a decrease of urinary oxalate.
  • reduction of urinary oxalate reduces or eliminate calcium oxalate formation and deposition in organs.
  • the subject is mammalian. In some embodiments, the subject is human. In some embodiments, the subject is cow, pig, monkey, sheep, dog, cat, fish, or poultry.
  • a guide RNAs comprising any one or more of the guide sequences in Table 1 or one or more sgRNAs from Table 2 (e.g., in a composition provided herein) is provided for the preparation of a medicament for treating a human subject having hyperoxaluria.
  • the guide RNAs, compositions, and formulations are administered intravenously. In some embodiments, the guide RNAs, compositions, and formulations are administered into the hepatic circulation.
  • a single administration of a composition comprising a guide RNA provided herein is sufficient to knock down expression of the mutant protein. In other embodiments, more than one administration of a composition comprising a guide RNA provided herein may be beneficial to maximize therapeutic effects.
  • treatment slows or halts hyperoxaluria disease progression.
  • treatment slows or halts progression of end stage renal disease (ESRD). In some embodiments, treatment slows or halts the need for kidney and/or liver transplant. In some embodiments, treatment results in improvement, stabilization, or slowing of change in symptoms of hyperoxaluria.
  • ESRD end stage renal disease
  • the invention comprises combination therapies comprising any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 (e.g., in a composition provided herein) together with an additional therapy suitable for alleviating hyperoxaluria and its symptoms, as described above.
  • the additional therapy for hyperoxaluria is vitamin B6, hydration, renal dialysis, or liver or kidney transplant.
  • the additional therapy is another agent that disrupts the LDHA gene, such as, for example, an siRNA directed to the LDHA gene.
  • the siRNA directed to the LDHA gene is DCR-PHXC.
  • the additional therapy is an agent that disrupts the HAO1 gene, such as, for example, an siRNA directed to the HAO1 gene.
  • the HAO1 siRNA is lumasiran (ALN-GO1; Alnylam).
  • the combination therapy comprises any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 together with a siRNA that targets HAO1 or LDHA.
  • the siRNA is any siRNA capable of further reducing or eliminating the expression of LDHA.
  • the siRNA is administered after any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 (e.g., in a composition provided herein). In some embodiments, the siRNA is administered on a regular basis following treatment with any of the gRNA compositions provided herein.
  • the combination therapy comprises any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 (e.g., in a composition provided herein) together with antisense nucleotide that targets LDHA.
  • the antisense nucleotide is any antisense nucleotide capable of further reducing or eliminating the expression of LDHA.
  • the antisense nucleotide is administered after any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 (e.g., in a composition provided herein).
  • the antisense nucleotide is administered on a regular basis following treatment with any of the gRNA compositions provided herein.
  • Lipid nanoparticles are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of the guide RNAs, compositions, or pharmaceutical formulations disclosed herein.
  • the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.
  • the invention comprises a method for delivering any one of the gRNAs disclosed herein to a subject, wherein the gRNA is associated with an LNP.
  • the gRNA/LNP is also associated with a Cas9 or an mRNA encoding Cas9.
  • the invention comprises a composition comprising any one of the gRNAs disclosed and an LNP.
  • the composition further comprises a Cas9 or an mRNA encoding Cas9.
  • the LNPs comprise cationic lipids.
  • the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid.
  • the LNPs comprise molar ratios of a cationic lipid amine to RNA phosphate (N:P) of about 4.5, 5.0, 5.5, 6.0, or 6.5.
  • N:P RNA phosphate
  • the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.
  • LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for treating a disease or disorder.
  • Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivery of any one of the gRNAs disclosed herein. In some embodiments, electroporation may be used to deliver any one of the gRNAs disclosed herein and Cas9 or an mRNA encoding Cas9.
  • the invention comprises a method for delivering any one of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNA is associated with an LNP or not associated with an LNP.
  • the gRNA/LNP or gRNA is also associated with a Cas9 or an mRNA encoding Cas9.
  • the guide RNA compositions described herein, alone or encoded on one or more vectors, are formulated in or administered via a lipid nanoparticle; see e.g., WO/2017/173054, filed Mar. 30, 2017 and published May 10, 2017 entitled “LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS COMPONENTS,” the contents of which are hereby incorporated by reference in their entirety.
  • the invention comprises DNA or RNA vectors encoding any of the guide RNAs comprising any one or more of the guide sequences described herein.
  • the vectors further comprise nucleic acids that do not encode guide RNAs.
  • Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding an RNA-guided DNA nuclease, which can be a nuclease such as Cas9.
  • the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA.
  • the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease, such as Cas9 or Cpf1.
  • the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas protein, such as, Cas9.
  • the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9).
  • the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system.
  • the nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA.
  • IVTT In Vitro Transcription
  • Spy Capped and polyadenylated Streptococcus pyogenes
  • Cas9 mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase.
  • Plasmid DNA containing a T7 promoter and a sequence for transcription was linearized by incubating at 37° C. to complete digestion with XbaI with the following conditions: 200 ng/4 plasmid, 2 U/4 XbaI (NEB), and 1 ⁇ reaction buffer.
  • the XbaI was inactivated by heating the reaction at 65° C. for 20 min.
  • the linearized plasmid was purified from enzyme and buffer salts using a silica maxi spin column (Epoch Life Sciences) and analyzed by agarose gel to confirm linearization.
  • the IVT reaction to generate Cas9 modified mRNA was incubated at 37° C. for 4 hours in the following conditions: 50 ng/4 linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U/ ⁇ L T7 RNA polymerase (NEB); 1 U/4 Murine RNase inhibitor (NEB); 0.004 U/4 Inorganic E.
  • ThermoFisher coli pyrophosphatase
  • ThermoFisher was added to a final concentration of 0.01 U/4, and the reaction was incubated for an additional 30 minutes to remove the DNA template.
  • the Cas9 mRNA was purified from enzyme and nucleotides using a MegaClear Transcription Clean-up kit according to the manufacturer's protocol (ThermoFisher). Alternatively, the Cas9 mRNA was purified with a LiCl precipitation method, which in some cases was followed by further purification by tangential flow filtration. The transcript concentration was determined by measuring the light absorbance at 260 nm (Nanodrop), and the transcript was analyzed by capillary electrophoresis by Bioanlayzer (Agilent).
  • the sequence for transcription of Cas9 mRNA used in the Examples comprised a sequence selected from SEQ ID NO: 501-515 as shown in Table 24.
  • RNA cargos e.g., Cas9 mRNA and sgRNA
  • the RNA cargos were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.
  • the LNPs used in Examples 2-4 contained ionizable lipid ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively.
  • the LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a ratio of gRNA to mRNA of 1:1 by weight.
  • the LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solutions and one volume of water.
  • the lipid in ethanol was mixed through a mixing cross with the two volumes of RNA solution.
  • a fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See WO2016010840 FIG. 2 .).
  • the LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v).
  • Diluted LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100 kD MWCO) and then buffer exchanged using PD-10 desalting columns (GE) into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS). The resulting mixture was then filtered using a 0.2 ⁇ m sterile filter. The final LNP was stored at 4° C. or ⁇ 80° C. until further use.
  • GE PD-10 desalting columns
  • Initial guide selection was performed in silico using a human reference genome (e.g., hg38) and user defined genomic regions of interest (e.g., LDHA protein coding exons), for identifying PAMs in the regions of interest. For each identified PAM, analyses were performed and statistics reported. gRNA molecules were further selected and rank-ordered based on a number of criteria known in the art (e.g., GC content, predicted on-target activity, and potential off-target activity).
  • a human reference genome e.g., hg38
  • user defined genomic regions of interest e.g., LDHA protein coding exons
  • a total of 84 guide RNAs were designed toward human LDHA (ENSG00000134333) targeting the protein exonic coding regions. Guides and corresponding genomic coordinates are provided above (Table 1). Forty of the guide RNAs have 100% homology with cynomolgus LDHA.
  • Additional guides were designed against a de novo Cynomolgus Macaque LDHA transcript.
  • Raw data were obtained from published transcriptome sequencing of liver sample from a female Mauritian-origin Cynomolgus Macaque (NCBI SRA ID: SRR1758956; Peng et al. (2015), Nucleic Acids Research, Volume 43, Issue D1, Pages D737-D742).
  • De novo transcriptome assembly was carried out using Trinity (v2.8.4; Grabherr et al. (2011), Nature Biotechnology, 29: 644-652) and SPAdes (v3.13.0; Bankevich et al. (2012), Journal of Computational Biology, 19:5).
  • PSH Primary human liver hepatocytes
  • PCH primary cynomolgus liver hepatocytes
  • Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat. 877272) at a density of 33,000 cells/well for PHH and 50,000 cells/well for PCH. Plated cells were allowed to settle and adhere for 5 hours in a tissue culture incubator at 37° C. and 5% CO 2 atmosphere. After incubation cells were checked for monolayer formation and were washed once with hepatocyte culture medium (Takara, Cat. Y20020 and/or Invitrogen, Cat. A1217601 and CM4000).
  • dgRNA ribonucleoprotein
  • RNP ribonucleoprotein
  • Cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) according to the manufacturer's protocol. Cells were transfected with an RNP containing Spy Cas9 (10 nM), individual guide (10 nM), tracer RNA (10 nM), Lipofectamine RNAiMAX (1.0 ⁇ L/well) and OptiMem.
  • RNP ribonucleoprotein
  • studies utilizing RNP transfection cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) according to the manufacturer's protocol. Cells were transfected with an RNP containing Spy Cas9 (10 nM), sgRNA (10 nM), Lipofectamine RNAiMAX (1.0 ⁇ L/well) and OptiMem.
  • LNPs Primary human and cyno hepatocytes were also treated with LNPs as further described below. Cells were incubated at 37° C., 5% CO 2 for 48 hours prior to treatment with LNPs. LNPs were incubated in media containing 3% cynomolgus serum at 37° C. for 10 minutes and administered to cells in amounts as further provided herein.
  • RNA cargos e.g., Cas9 mRNA and gRNA
  • N:P lipid amine to RNA phosphate
  • PHH and PCH transfected cells were harvested post-transfection at 72 or 96 hours.
  • the gDNA was extracted from each well of a 96-well plate using 50 ⁇ L/well BuccalAmp DNA Extraction solution (Epicentre, Cat. QE09050) according to manufacturer's protocol. All DNA samples were subjected to PCR and subsequent NGS analysis, as described herein.
  • PCR primers were designed around the target site within the gene of interest (e.g. LDHA), and the genomic area of interest was amplified. Primer sequence design was done as is standard in the field.
  • PCR was performed according to the manufacturer's protocols (Illumina) to add chemistry for sequencing.
  • the amplicons were sequenced on an Illumina MiSeq instrument.
  • the reads were aligned to the reference genome (e.g., hg38) after eliminating those having low quality scores.
  • the resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion or deletion (“indel”) was calculated.
  • the editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of sequence reads with insertions or deletions (“indels”) over the total number of sequence reads, including wild type.
  • LDHA Lactate Dehydrogenase A
  • LNPs Primary human hepatocytes were treated with LNP formulated with select guides from Table 1 as further described in Example 3. LNPs were incubated in media (Takara, Cat. Y20020) containing 3% cynomolgus serum at 37° C. for 10 minutes. Post-incubation the LNPs were added to the human hepatocytes. Twenty-one days post-transfection, the media was removed and the cells were lysed with 50 ⁇ L/well RIPA buffer (Boston Bio Products, Cat. BP-115) plus freshly added protease inhibitor mixture consisting of complete protease inhibitor cocktail (Sigma, Cat. 11697498001), 1 mM DTT, and 250 U/ml Benzonase (EMD Millipore, Cat.
  • AGT-deficient mice were treated with LNP formulated with select guides as further described in Example 4. Livers were harvested from the mice post-treatment and 60 mg portions were used for protein extraction. The samples were placed in bead tubes (MP Biomedical, Cat. 6925-500) and lysed with 600 ⁇ L/sample of RIPA buffer (Boston Bio Products, Cat. BP-115) plus freshly added protease inhibitor mixture consisting of complete protease inhibitor cocktail (Sigma, Cat. 116974500) and homogenized at 5.0 m/sec. The samples were then centrifuged at 14,000 RPM for 10 min. at 4° C. and the liquid was transferred to a new tube. A final centrifugation was performed at 14,000 RPM for 10 min. and the samples were quantified using a Bradford assay as described above.
  • a western blot was performed to assess LDHA protein levels. Lysates were mixed with Laemmli buffer and denatured at 95° C. for 10 minutes. The blot was run using the NuPage system on 10% Bis-Tris gels (Thermo Fisher Scientific, Cat. NP0302BOX) according to the manufacturer's protocol followed by wet transfer onto 0.45 ⁇ m nitrocellulose membrane (Bio-Rad, Cat. 1620115). After the transfer membrane was rinsed thoroughly with water and stained with Ponceau S solution (Boston Bio Products, Cat. ST-180) to confirm complete and even transfer. The blot was blocked using 5% Dry Milk in TBS for 30 minutes on a lab rocker at room temperature.
  • the blot was rinsed with TBST and probed with rabbit ⁇ -LDHA polyclonal antibody (Sigma, Cat. SAB2108638 for cell lysate or Genetex, Cat. GTX101416 for mouse liver lysate) at 1:1000 in TBST.
  • rabbit ⁇ -LDHA polyclonal antibody Sigma, Cat. SAB2108638 for cell lysate or Genetex, Cat. GTX101416 for mouse liver lysate
  • beta-actin was used as a loading control (Novus, Cat. NB600-501) at 1:1000 in TBST and incubated simultaneously with the LDHA primary antibody.
  • GAPDH was used as a loading control (Abcam, ab8245) at 1:1000 in TBST and incubated simultaneously with the LDHA primary antibody.
  • the blot was sealed in a bag and kept overnight at 4° C. on a lab rocker. After incubation, the blot was rinsed 3 times for 5 minutes each in TBST and probed with secondary antibodies to Mouse and Rabbit (Thermo Fisher Scientific, Cat. PI35518 and PISA535571) at 1:12,500 each in TBST for 30 minutes at room temperature. After incubation, the blot was rinsed 3 times for 5 minutes each in TBST and 2 times with PBS. The blot was visualized and analyzed using a Licor Odyssey system.
  • LDHA Lactate Dehydrogenase A
  • a biochemical method (e.g., Wood K D et al., Biochim Biophys Acta Mol Basis Dis. 2019 Sep. 1; 1865(9):2203-2209; PMC6613992) was used for lactate dehydrogenase activity.
  • tissue was homogenized in iced cold lysis buffer (25 mM HEPES, pH 7.3, 0.1% Triton-X-100) with probe sonication to give a 10% wt/vol lysate.
  • LDH activity was measured by the increased in absorbance at 340 nm with the reduction of NAD to NADH in the presence of lactate.
  • Lactate to pyruvate activity of LDG was measured with 20 mM lactate, 100 mM Tris-HCL, pH 9.0, 2 mM NAD+, 0.01% liver lysate.
  • a Cooomassie Plus protein assay kit (Pierce, Rockford, Ill.), with bovine serum albumin (BSA) as the standard, was used to determine protein concentration in tissue lysates.
  • part of the urine collection was acidified to pH between 1 and 2 with HCl prior to storage at ⁇ 80° C. to prevent any possible oxalate crystallization that could occur with cold storage and/or oxalogensis associated with alkalinization.
  • the remaining nonacidified urine was frozen at ⁇ 80° C. for the measurement of creatinine.
  • Plasma preparations were filtered through Nano-sep centrifugal filters (VWR International, Batavia, Ill.) with a 10,000 nominal molecular weight limit to remove macromolecules prior to ion chromatography coupled with mass spectrometry or ICMS (Thermo Fisher Scientific Inc., Waltham, Mass.).
  • Centrifugal filters were washed with 10 mM HCl prior to sample filtration to remove any contaminating trace organic acids trapped in the filter device.
  • Liver tissue was extracted with 10% (wt/vol) trichloroacetic acid (TCA) for organic acid analysis.
  • TCA trichloroacetic acid
  • These organic acids were measured by ICMS following removal of TCA by vigorous vortexing with an equal volume of 1,1,2-trichlorotrifluoroethane (Freon)-trioctylamine (3:1, vol/vol; Aldrich, Milwaukee, Wis.), centrifuging at 4° C. to promote phase separation, and collecting the upper aqueous layer for analysis.
  • Urinary creatinine was measured on a chemical analyzer, and urinary oxalate by ICMS, as previously described.
  • Selected-ion monitoring at the following mass/charge ratios and cone voltages were used to quantify lactate (SIM 89.0, 35 V) and 13C3-lactate (SIM 92.0, 35 V). Pyruviate was measured by IC/MS with an AS11-HC 4 ⁇ m, 2 ⁇ 150 mm, anion exchange column at a controlled temperature of 30° C. and a DionexTM ERSTM 500 anion electrolytically regenerated suppressor. A gradient of KOH from 0.5 to 80 mM over 60 min at a flow rate of 0.38 ml/min was used to separate sample anions. The mass spectrometer (MSQ-PLUS) was operated in ESI negative mode, needle voltage 1.5 V, 500° C.
  • a biochemical method See, e.g., Cameron et al., Nature Methods. 6, 600-606; 2017 was used to determine potential off-target genomic sites cleaved by Cas9 targeting LDHA.
  • 10 modified sgRNA targeting human LDHA and two control guides with known off-target profiles were screened using isolated HEK293 genomic DNA and the potential off-target results were plotted in FIG. 1 .
  • the assay identified potential off-target sites for the sgRNAs tested.
  • a large number of potential off-target sites are typically recovered, by design, so as to “cast a wide net” for potential sites that can be validated in other contexts, e.g., in a primary cell of interest.
  • the biochemical method typically overrepresents the number of potential off-target sites as the assay utilizes purified high molecular weight genomic DNA free of the cell environment and is dependent on the dose of Cas9 RNP used. Accordingly, potential off-target sites identified by these methods may be validated using targeted sequencing of the identified potential off-target sites.
  • primary hepatocytes are treated with LNPs comprising Cas9 mRNA and a sgRNA of interest (e.g., a sgRNA having potential off-target sites for evaluation).
  • LNPs comprising Cas9 mRNA and a sgRNA of interest (e.g., a sgRNA having potential off-target sites for evaluation).
  • the primary hepatocytes are then lysed and primers flanking the potential off-target site(s) are used to generate an amplicon for NGS analysis. Identification of indels at a certain level may validate potential off-target site, whereas the lack of indels found at the potential off-target site may indicate a false positive in the off-target assay that was utilized.
  • Lipid Nanoparticle Formulations Containing Spy Cas9 mRNA and sgRNA in Primary Human and Cynomolgus Hepatocytes
  • Lipid nanoparticle (LNP) formulations of modified sgRNAs targeting human LDHA and those homologous in cyno were tested on primary human hepatocytes and primary cynomolgus hepatocytes in a dose response assay.
  • the LNPs were formulated as described in Example 1.
  • Primary human and cynomolgus hepatocytes were plated as described in Example 1. Both cell lines were incubated at 37° C., 5% CO 2 for 48 hours prior to treatment with LNPs. LNPs were incubated in media containing 6% cynomolgus serum at 37° C. for 10 minutes.
  • the LNPs were added to the human or cynomolgus hepatocytes in an 8 point 3-fold dose response curve starting at 300 ng Cas9 mRNA.
  • the cells were lysed 96 hours post-treatment for NGS analysis as described in Example 1.
  • the dose response curve data for the guide sequences in both cell lines is shown in FIGS. 2 and 3 .
  • the percent editing at the 22 nM concentration are listed below in Tables 9 and 10.
  • Table 9 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested LDHA sgRNAs at 22 nM delivered with Spy Cas9 via LNP in primary human hepatocytes. These samples were generated in duplicate.
  • Table 10 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested LDHA sgRNAs at 22 nM delivered with Spy Cas9 via LNP in primary cynomolgus hepatocytes. These samples were generated in triplicate.
  • Lipid nanoparticle (LNP) formulations of modified sgRNAs targeting human LDHA were administered to primary human hepatocytes to generate samples for Western Blotting.
  • the LNPs were formulated as described in Example 1.
  • Primary human hepatocytes were plated as described in Example 1. Cells were incubated at 37° C., 5% CO 2 for 48 hours prior to treatment with LNPs. LNPs were incubated in media containing 6% cynomolgus serum at 37° C. for 10 minutes. Post-incubation the LNPs were added to the human hepatocytes at a concentration of 25 nM of sgRNA per sample.
  • NGS sequencing a portion of the cells were collected and processed for NGS sequencing as described in Example 1. The remaining cells were harvested twenty-one days post-transfection and whole cell extracts (WCEs) were prepared and subjected to analysis by Western Blot as described in Example 1.
  • the editing data for these cells is provided in Table 12.
  • WCEs were analyzed by Western Blot for reduction of LDHA protein.
  • Full length LDHA protein has 332 amino acids and a predicted molecular weight of 36.6 kD. A band at this molecular weight was observed in the control lane (untreated cells) but not in any of the treated lanes ( FIG. 4 ).
  • sgRNAs targeting LDHA were administered to primary human and cynomolgus hepatocytes by lipofection to generate samples for qPCR.
  • the lipofection samples were formulated as described in Example 1.
  • Primary hepatocytes were plated as described in Example 1. Cells were incubated at 37° C., 5% CO 2 for 48 hours prior to treatment with lipid packets. Lipofection samples were incubated in media containing 6% cynomolgus serum at 37° C. for 10 minutes. Post-incubation the lipid packets were added to the hepatocytes at multiples concentrations. At 96 hours post-lipofection, the cells were collected and processed for RNA as described in Example 1. Average LDHA transcript reduction in primary human and cynomolgus hepatocytes at 15 nM guide is contained within Table 13 below, with full dose-response data displayed in FIGS. 13A-13B .
  • mice Both wildtype and AGT-deficient mice (Agxt 1 ⁇ / ⁇ ), e.g., null mutant mice lacking liver AGXT mRNA and protein were used in this study.
  • the AGT-deficient mice exhibit hyperoxaluria and crystalluria and thus represent a phenotypic model of PH1, as previously described by Salido et al., Proc Natl Acad Sci USA. 2006 Nov. 28; 103(48):18249-54.
  • the wildtype mice were used to determine which formulation to test in the AGT-deficient mice.
  • RNPs comprising dgRNAs targeting murine Ldha were screened for editing efficiency similarly as described in Example 2 for the human and cyno LDHA-targeting gRNAs. Having identified active gRNAs from the dgRNA screen, a smaller set of modified sgRNAs based on these gRNAs were synthesized for further evaluation in vivo.
  • LNPs containing modified sgRNAs targeting murine Ldha were dosed via the lateral tail vein in a volume of 0.2 mL per animal (approximately 10 mL per kilogram body weight).
  • the LNPs were formulated as described in Example 1.
  • LNP containing G009439 was administered to the AGT-deficient mice in a dose response (0, 0.25, 0.5, 1, and 2 mpk) with respect to total mRNA cargo. These mice were housed in metabolic cages and urine was collected at various time points for oxalate levels, e.g., as described by Liebow et al., J Am Soc Nephrol. 2017 February; 28(2):494-503. Editing of the Ldha gene and secretion of oxalate were shown to increase and decrease, respectively, with increasing doses of LNP. The % editing and ug urinary oxalate/mg creatinine excreted are contained within Table 15 below and displayed in FIGS. 14A-14C .
  • the percent editing in each treated animal is reported in Table 16, and the % reduction of urinary oxalate is shown at each week post-treatment in Table 18.
  • a sgRNA G000723 which targets murine Hao1.
  • oxalate levels were reduced one week following treatment with LNP comprising this gRNA and this level of reduction was sustained out to at least 5 weeks post-dose.
  • LDHA protein in treated and nontreated mice was additionally characterized through immunohistochemical staining as described in Example 1 and depicted in FIG. 8 .
  • a progressive reduction in LDHA staining was observed in 0.3 mpk-dosed mice and 1 mpk-dosed mice compared to control mice.
  • FIG. 9 shows a correlation with an R 2 value of 0.95 between the editing and protein levels in Table 19.
  • liver samples from mice treated with 1 mpk of Ldha LNP were processed for LDH activity as described in Example 1. Reduction of LDH activity was observed in liver samples from mice treated with 1 mpk of Ldha LNP. Specific activity ( ⁇ mol/min/mg protein) from the treated and control mice are contained in Table 21 below and data displayed in FIGS. 15A-15B .
  • mice with compromised kidney function were obtained from the Jackson Laboratory (Bar Harbor, Me.).
  • animals were bled for baseline lactate levels as described in Example 1. Animals were then dosed with LNP containing G009439 at a dose of 2 mpk (n 6).
  • lactate challenge comprising of 2 g/kg of sodium lactate dissolved in phosphate buffered saline (concentration 200 mg/mL, ⁇ 18 mM) pH 7.4, delivered intraperitoneally. Animals were tail-bled before the challenge and then 15, 30, 60, and 180 minutes post-challenge. Blood samples were analyzed for lactate levels as described in Example 1. No significant differences in lactate clearance were observed in mice that had received the nephrectomy surgeries and LDHA LNP, compared to sham surgery and vehicle treatment mice. Table 23 below details the average plasma pyruvate across animal groups, as also shown in FIG. 17 .

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EP3856901A1 (fr) 2021-08-04
TW202028460A (zh) 2020-08-01
JP2022502055A (ja) 2022-01-11
SG11202102660RA (en) 2021-04-29
IL281529A (en) 2021-05-31
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PH12021550686A1 (en) 2022-02-28
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